UNIVERSITÀ DEGLI STUDI DI NAPOLI FEDERICO II FACOLTÀ DI MEDICINA E CHIRURGIA SCUOLA DI DOTTORATO IN MEDICINA MOLECOLARE DOTTORATO DI RICERCA IN GENETICA E MEDICINA MOLECOLARE XXII CICLO TESI DI DOTTORATO “Epigenetic dynamics of Helicobacter pylori-induced COX-2 activation” Coordinatore Candidata Prof. Roberto Di Lauro Silvia PELUSO ANNO 2009 UNIVERSITÀ DEGLI STUDI DI NAPOLI FEDERICO II DIPARTIMENTO DI BIOLOGIA E PATOLOGIA CELLULARE E MOLECOLARE “LUIGI CALIFANO” SCUOLA DI DOTTORATO IN MEDICINA MOLECOLARE DOTTORATO DI RICERCA IN GENETICA E MEDICINA MOLECOLARE XXII CICLO Tesi di Dottorato “Epigenetic dynamics of Helicobacter pylori-induced COX-2 activation” Docente Guida Prof. Lorenzo Chiariotti 2 INDEX INTRODUCTION pag. 4 HISTONE MODIFICATION 8 Acethylation 10 Methylation 12 Ubiquitination 14 Biotinilation 15 DNA METHYLATION 16 GENE EXPRESSION AND SILENCING 19 HELICOBACTER PYLORI 29 Induction of enzymes and cytokines by H. pylori 36 AIM OF THIS STUDY 40 MATERIALS AND METHODS 41 Bacterial strain and growth condition 41 Human gastric epithelial cells in colture 42 Preparation of cell extracts and western blot analysis 42 Real-time PCR 43 HDAC activity assay 45 Chromatin immunoprecipitation (ChIP) assay 46 Quantification of ChIP DNA by real-time PCR 48 Methylation DNA analysis with MALDI-TOF MS 49 Statistical analysis 50 RESULTS 51 Helicobacter pylori infection induces activation of COX-2 gene 51 Role of Histone acetylation on H. pylori- induced COX-2 expression 53 Heffect of H. pylori on the recruitment of HDAC-1 and HDAC-2 to the COX-2 promoter 58 Histone methylation at H. pylori induced COX-2 60 CpG methylation of COX-2 promoter 62 Nf-κB associates with COX-2 promoter upon H. pylori infection 64 DISCUSSION 66 ACKNOWLEDGEMENTS 77 REFERENCES 78 3 INTRODUCTION The eukaryotic genome is assembled as a nucleoprotein complex known as chromatin which is a structural polymer consisting of positively charged histone proteins in addition to DNA. It provides a dynamic platform that controls all DNA-mediated processes within the nucleus. The basic unit of chromatin is the nucleosome that consists of 147 bp of DNA wrapping nearly twice around the octamer, containing two copies of each of core histones H2A, H2B, H3 and H4. Each nucleosome is separated by 10-16 bp linker DNA and this bead on a string arrangement constitutes a chromatin fiber of ~10 nm in diameter. Each core histone within the nucleosome contains a globular domain, which mediates histone-histone interactions and also bears a highly dynamic amino terminal tail approximately 20-35 residues in length and is rich in basic amino acids. These tails extend from the surface of the nucleosome. Histone H2A also has an additional ~37 amino acid, carboxy-terminal domain protruding from the nucleosome. All histone proteins are modified inside the nucleus of the cell, but so far only a few modifications have been studied. A fundamental tenet of biochemistry is that proteins are composed of 20 basic building blocks. Given the limited range of chemical functionality present in the amino acid side chains, the diversity of protein structure and function is truly extraordinary. In reality, the situation is not very complex: nature uses several modifications of proteins to complement and expand its chemical repertoire. In fact, many distinct posttranslational modifications (PTMs) have been identified to date, and the number and variety of modifications increase continuously. Histones are subject to many PTMs including acetylation and methylation of lysines (K) and arginines (R), phosphorylation of serine (S) and threonines (T), ubiquitination, sumolyation and biotinylation of lysines as well as ADP ribosylation. These modifications are recognized by specific protein-protein modules and can regulate each other as well (Yap and Zhou, 2006). Recent findings have revealed that these histone tails do not contribute to the structure of individual nucleosomes or to their stability but play an important role in folding of nucleosomal arrays into higher order chromatin structure (Paterson and Laniel, 2004). Eukaryotes have developed many histone-based strategies to introduce variation into the chromatin fiber because the chromatin is the physiological template for all DNA-mediated processes, and 5 these strategies are likely to control the structure and/or function of the chromatin fiber. Two of the most common strategies employed are the PTMs of histones and replacement of major histone species by the variant isoforms (Taverna et al., 2006). Many studies have shown that the site-specific combinations of histone modifications correlate well with particular biological functions, such as transcription, silencing, heterochromatization, DNA repair and replication. These observations have led to the idea of a ‘histone code’, although the degree of specificity of these codes may vary as particular combinations of histone marks do not always dictate the same biological function. Moreover, there is a clear indication that mistakes in PTMs may be involved in many human diseases, especially cancer (Somech et al., 2004). 6 Figura 1 Covalent marks on chromatin: Chromatin consists of repeated units of 146 bp of DNA wrapped 1.7 times around a octamer composed of two copies each of the four core histones; H2a, H2b, H3 and H4. Chromatin provides a structural platform that is subject to extensive post- translational modification. These include methylation, acetylation, phosphorylation and ubiquitination of specific histone residues; methylation of CpG dinucleotides; exchange of histones (a); changes in the relative position of the nucleosome mediated by ATP-dependent remodelling complexes (b); induction of double-stranded DNA breaks by topoisomerase II (c) and the generation of single-stranded DNA breaks by topoisomerase I (d). 7 HISTONE MODIFICATIONS More than 35 years ago, it was noticed that transcriptionally active histones are acetylated in vivo, and this led to the speculation that covalent modifications of histones might have a role in determining the states of gene activity (Allfrey et al., 1964). The histone N-terminal tails, exposed on the nucleosome surface, are subject to a variety of enzyme- catalyzed PTMs. As already mentioned, several histone-tail modifications have been identified. Moreover, each lysine residue can accept one, two or even three methyl groups, and similarly an arginine can be either mono- or dimethylated, which adds to the complexity. Although Strahl and Allis (2000) proposed the histone code, it’s not clear how the code is established and maintained till now. Many modification sites are close enough to each other and it seems that modification of histone tails by one enzyme might influence the rate and efficiency at which other enzymes use the newly modified tails as a substrate (Chung, 2002). The model proposed by Allis has set a ground for researchers to discover post-translational writers, the enzymes that modify the histones post translationally, and also the readers, the modules or motifs that recognize the 8 histones based on their modification state (Strahl and Allis, 2000). Although the basic composition of the nucleosome is the same over long stretches of chromatin, the specific pattern of modifications on the nucleosome creates local structural and functional diversity, delimiting chromatin subdomains. Therefore, the challenge of histone PTMs has shifted from identifying sites of modification to identifying combinations of histone modification patterns that dictate specific biological readouts. Currently, the histone code is under a lot of investigation and is gaining experimental support as well. Methylation of DNA at cytosine residues as well as PTMs of histones, including phosphorylation, acetylation, methylation and ubiquitylation, contributes to the epigenetic information carried by chromatin. These changes play an important role in the regulation of gene expression by modulating the access of regulatory factors to the DNA. The use of a combination of biochemical, genetic and structural approaches has been allowed to demonstrate the role of chromatin structure in transcriptional control. The structure of nucleosomes has been elucidated and enzymes involved in DNA or histone modifications have been extensively characterized. Because deregulation of epigenetic marks has been reported in many 9 cancers, a better understanding of the underlying molecular mechanisms bears the promise that new drug targets may soon be found. Acetylation Although it has been known for many years that histones in eukaryotes are modified by acetylation, it is only in the past decade that the role of histone acetylation in transcription regulation has been focused (Grunstein 1997, Kou and Allis, 1998). Of all the histone modifications, acetylation is the most studied one. In the beginning, many of the enzymes responsible for acetylation of histones were known as transcriptional co-activators and later as enzymes. During specific biological processes, selected lysines such as lysine 9 and 14 are acetylated. Acetylation of lysine residues at core histone N-terminal is achieved through enzymes called histone acetyltranferases (HATs). However, the steady state balance of this modification is achieved using the orchestrated action of HATs and one more species of enzymes, namely histone deacetylases (HDACs) (Brownell and Allis, 1996; Kuo and Alis, 1998; Roth et al., 2000). There are two different types of HATs: type A-HATs, which are responsible for the acetylation of histones and are 10 directly involved